Project Summary Roles of Cholesterol and Membrane Nanodomains in the Amyloidogenic Pathway Cholesterol is thought to play a key regulatory role in the formation of the amyloid-? polypeptide (A?) via the amyloidogenic pathway in which the full length amyloid precursor protein is cleaved by ?-secretase to release is transmembrane 99 residue C-terminal domain (C99). C99 is then cleaved by ?-secretase to release A?. Membrane cholesterol appears to promote the amyloidogenic pathway, with much evidence pointing to a mechanism involving cholesterol-enriched regions of the plasma membrane often referred to as lipid rafts. According to this model, cholesterol promotes partitioning both of APP and C99 into lipid rafts, which are enriched in ?-secretase and ?-secretase, thereby activating amyloidogenic cleavage. Rafts are therefore proposed to spatially segregate the amyloidogenic pathway from the bulk membrane phase. However, much of the evidence in support of this model is indirect. It is the premise of this work that it is important to test this model which, if correct, suggests a strategy for reducing A? formation by reducing raft partitioning of APP/C99. On the other hand, if the model is incorrect, the existing literature supporting this model may need to be re- assessed and the pursuit of therapeutic strategies based on this model would possibly need to be viewed as a red herring. A sub-premise of this work is that it is critical to conduct studies of the biophysical and structural biological basis for partitioning of C99/APP between raft and non-raft phases. These studies will provide fundamental insight how major changes in membrane phase properties impact membrane protein structure and localization. Moreover, a molecular understanding of the basis for the phase partitioning preferences of APP and C99 may inspire rational pharmacological strategies for altering the phase preferences of these molecules for the purpose of reducing A? formation. We note that in order to maintain an appropriate (feasible!) scope for the studies to be carried out, we will focus on ?-secretase and C99. Studies of full length APP and ?-secretase represent a future direction for this work after the completion of the aims below. Aim 1. Test the hypothesis that C99 and ?-secretase preferentially localize to membrane raft domains. Aim 2. Test the hypothesis that raft-association alters the structure of C99. Aim 3. Determine how the cleavage of C99 by ?-secretase is modulated by membrane partitioning, membrane fluidity, C99 structure, and C99-cholesterol complex formation.